How does the General Theory of Cardiac Chronotropy and Inotropy Apply to Aging? Both our earlier and very recent studies have contributed seminal perspectives toward understanding age-associated deterioration of both cardiac contractility and heart rate in both humans, and in animal models. Our studies of healthy participants in the Baltimore Longitudinal Study of Aging (BLSA) demonstrated substantial age-associated changes in the ability to increase heart rate and reduce end systolic volume in response to graded exercise stress. (Of note, only the latter, but not the former, can be improved by physical conditioning.) We subsequently demonstrated that deficits both in contractility and heart rate in humans are due, in part, to reduced beta-AR stimulation response of cAMP-PKA. 1. Contractility In isolated rat cardiac ventricular muscle, we had directly demonstrated a reduction in the Ca2+ cycling and contractile response to beta-AR stimulation. The relaxation time of the Ca2+ transient and contraction in the absence of beta-AR stimulation were prolonged, which we traced to a reduced expression of SERCA2. The AP was also markedly prolonged, due to age-associated changes in L-type Ca2+ and K+ currents. In single VM, in addition to numerous properties of surface membrane ion channels, we documented an age-associated reduction in the VM Ca2+ clock, manifest as a prolonged time for restitution of the excitation- Ca2+ release-contraction coupling process, was due to a prolonged restitution time for SR Ca2+ release via RyRs in response to activation by an L-type Ca2+ current. We also showed that SR Ca2+ loading and the amplitude of the Ca2+ transient were preserved in myocytes from the old heart, by virtue of the prolonged AP. 2. Heart rate Our discovery of heart rate regulation by a coupled-clock system in rabbit SANC enabled progress, finally (after 25 years), on the elucidation of potential cellular mechanisms of the age-associated reduction in chronotropic reserve. Specifically, our conceptual breakthrough that regulation of basal pacemaker cell automaticity requires Ca-PKA-CaMKII signaling regulated by PDE and phosphatase activities, and that stimulation of receptors merely extended this regulation, led us to hypothesize that mechanisms involved in the deterioration of mechanisms that regulate of intrinsic heart rate that accompanies advancing age and that those regulating heart rate reserve may involve a slowing of the Ca2+ clock of SANC and linked, in some respects, at least, to the age-associated deterioration of mechanisms that regulate Ca2+ cycling and contractility in VM. In sinoatrial tissue under basal state the beating rate is lower in aged mice, but the rhythm variability is higher. The maximal responses of sinoatrial tissue beating rate and rhythm variability to autonomic receptor stimulation remain intact in mice, but both are less sensitive in advanced age. Changes in beating rate in response to autonomic receptor stimulation occur concurrently with changes in rhythm variability; i.e., increase in beating rate is associated with decrease in rhythm variability and vice versa. In isolate single SANC from young, old C57B and transgenic mice we tested our hypothesis that changes in spontaneous sarcoplasmic reticulum (SR) Ca2+ cycling and SANC ability to response to the cAMP-PKA-dependent signaling might play a significant role in the age-related SAN dysfunction and reduced beating rate with age. We found that the spontaneous AP firing rate of single SANC declines with age; the maximum Ca2+ release flux of single SANC in response to an AP declines with age and the kinetics of relaxation of the Ca2+ transient become prolonged with age. We measured spontaneous local Ca2+ releases (LCRs) in permeabilized SANC from young and old C57B mice at constant free Ca2. We discovered a reduced SR Ca2+ load, smaller size, duration and number of LCR events in old vs young SANC Moreover, when we activated the cAMP-PKA-Ca2+ pathway by phosphodiesterase inhibitor (IBMX) we found an increase in average LCR characteristics (size, duration, amplitude) and periodicity in young vs old. Reanalyzing data with the novel auto spark detection program showed an increase in LCR Ca2+ signal in response to PDE inhibition in young SANC but not old SANC, confirming our hypothesis that sensitivity to PDE inhibition is reduced by age. PLB phosphorylation at Ser16 immunolabeling in control conditions did not differ with age, but increased 2.2 fold after incubation with 10M IBMX in young but not old SANC. We conclude that intrinsic SR Ca2+cycling and its response to PDE inhibition decline with age, and that this may explain why the aged heart cannot beat as fast as the young When we tested RyRs, SERCA, PLB by Western blots we found that total PLB and P-PLB (Ser-16) levels were not significantly different between the young and old SAN tissues. However, RyRs and SERCA proteins were less abundant in old than in young mouse hearts. Specifically, the SAN, but not atrial and ventricular tissues, displayed a significant decrease in the amount of RyRs and SERCA proteins in old mice compared to young mice. Our Western blot data also showed that NCX protein level was significantly lower in old than in young SAN tissue. The relative contributions of age changes in autonomic input to SAN and intrinsic SAN functions to age-associated changes in heart rate (HR) or HR variability (HRV) are unknown. We recorded HR and HRV in vivo in aged (24 months) and adult (3 months) C57BL/6 male mice via EKG during light anesthesia in the basal state, and in the presence of 0.5mg/mlAtropine+1mg/ml Propranolol (intrinsic conditions). We recorded the intrinsic beating rate (BR) and BR variability (BRV) (in the absence of autonomic nerve input) in intact isolated SAN via a surface electrogram. While basal HR did not significantly change with age, the intrinsic HR in vivo and the intrinsic BR in intact isolated SAN were lower in advanced age. Therefore, an age-associated increase in sympatatic autonomic input to the SAN maintains a normal basal HR. While basal HRV, assessed in the time-domain (coefficient of variance) did not significantly vary with age, intrinsic HRV in vivo and BRV ex vivo increased in the aged mice compared to adult. Therefore, pacemaker functions intrinsic to the SAN become more variable in advanced age, and this increase in intrinsic variability is compensated for by autonomic nervous input. Spectral analyses uncovered a similar age-associated increase in the low frequency regime power (0.34<f<1.27Hz) of both the basal and intrinsic HR in vivo and BR ex vivo. There were no significant age-associated changes in very low or high frequencies. Therefore, HRV not only reports variability in autonomic nerve input to the heart. Similar age-associated changes in HRV both in vivo and ex vivo indicate that the age-associated changes in HRV are determined, in part at least, by age-associated changes in intrinsic pacemaker function. Therefore, age differences in HRV are not solely due to differences in autonomic input to the SAN. Our results suggest that the intrinsic cAMP-PKA-Ca2+ signaling is deficient in the aged mice. This deficiency may result from: 1) reduced amount or function of Ca2+ cycling proteins e.g. SR Ca2+ pump, PLB, RyRs. and/or 2) reduced phosphorylation of Ca2+ cycling proteins e.g. PLB, in response to an increase in cAMP-mediated-PKA dependent phosphorylation.